The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. endotoxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. endotoxin delivery vehicles (Gaertner and Kim, 1988). Thus, isolated B.t. endotoxin genes are becoming commercially valuable.
Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis var. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystal called a .delta.-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
In recent years, however, investigators have discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely B.t. var. israelensis and B.t. var. tenebrionis (a.k.a. M-7, a.k.a. B.t. var. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, 1989). See also Couch, 1980 and Beegle, 1978. Krieg et al., 1983, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and the beetle Agelastica alni.
Recently, new subspecies of B.t. have been identified, and genes responsible for active .delta.-endotoxin proteins have been isolated (Hofte and Whiteley, 1989). Hofte and Whiteley classified B.t. crystal protein genes into 4 major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryII (Coleoptera-specific), and CryIII (Diptera-specific). Prefontaine et al., 1987, describe probes useful in classifying lepidopteran-active genes. The discovery of strains specifically toxic to other pests has been reported (Feitelson et al., 1992).
B.t. crystalline toxins are generally recognized as being protoxins, requiring either particular physicochemical conditions (i.e., pH, redox, ionic strength), or the action of certain proteases, or both, to generate an active toxin (Hofte and Whiteley, 1989). In most cases, the insect supplies conditions for activation of the toxin; however, cases have been documented where pre-solubilization or pre-proteolysis have been necessary for optimum activity (Jacquet el al., 1987) or detection of activity (Hofte et al., 1992).
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf and Whiteley, 1981). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclose the expression of B.t. crystal proteins in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis (a.k.a. B.t. san diego, a.k.a. M-7) which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses Bacillus thuringiensis var. israelensis toxins which are active against dipteran pests and reports that a protein of about 27 kD, and fragments thereof, are responsible for the dipteran activity. U.S. Pat. No. 4,849,217 discloses B.t. isolates which have activity against the alfalfa weevil. U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art.
The alfalfa weevil, Hypera postica, and the closely related Egyptian alfalfa weevil, Hypera brunneipennis, are the most important insect pests of alfalfa grown in the U.S. with 2.9 million acres infested in 1984. An annual sum of 20 million dollars is spent to control these pests. The Egyptian alfalfa weevil is the predominant species in the southwestern U.S., where it undergoes aestivation (i.e., hibernation) during the hot summer months. In all other respects, it is identical to the alfalfa weevil, which predominates throughout the rest of the U.S.
The larval stage is the most damaging in the weevil life cycle. By feeding at the alfalfa plants growing tips, the larvae cause skeletonization of leaves, stunting, reduced plant growth, and, ultimately, reductions in yield. Severe infestations can ruin an entire cutting of hay. The adults, also foliar feeders, cause additional, but less significant, damage.
The rice water weevil, Lissorhoptrus aryzophilus, is a major insect pest of rice in North America and Southeast Asia. See Smith, M. C. (1983) "The Rice Water Weevil, Lissorhoptrus oryzophilus Kuschel," Exotic Plant Quarantine Pests and Possibilities for Introduction of Plant Materials, pp. 3-9. The rice water weevil can be directly responsible for average yield reductions of 10% or more if not treated with the proper insecticides. Rice water weevil larvae cause significant damage to the root systems of cultivated rice. Adult rice water weevils are small, black, oblong weevils (2.8-3.2 mm long.times.1.2-1.8 mm wide) with gray scales. Adults feed on rice by rasping away the leaf epidermis leaving skeletonized longitudinal slits on the upper leaf blades. Adults weevils appear to prefer two-week old rice plants over those of seven-week old plants and increased levels of nitrogen fertilizers increase the level of feeding. Adults also feed on individual grains of headed rice consuming the floral part or the endosperm of the developing rice kernel. Weevils enter a true diapause, fly to hibernation sites as early as July and overwinter in bunch grasses, Spanish moss and ground trash. Upon emerging in the spring the weevils migrate to flooded rice fields where they mate. Eggs are deposited in submerged leaf sheaths on the lower part of the rice plant and hatch within 4-9 days.
The rice water weevil larvae have 4 instars. The length of each instar phase is temperature dependent and under normal field conditions the larval stages last about 27 days. The larvae have paired dorsal tracheal hooks which function as modified spiracles. The apical segment of the hook is heavily sclerotized and is used to pierce root tissue and sequester air. This allows the larvae to live below the water surface. The pupae form in an oval, water-tight mud cell and resembles the adult in size and shape but is white in color. The duration of the pupal stage is seven days at 27.degree. C. Two and three generations per season have been reported.
Control of the rice water weevil is difficult due to its terrestrial, aquatic and soil habitats. Chemical insecticides have been used in the past with limited success. Native resistant rice cultivars are being sought with only low to moderate resistant lines being discovered.
The International Rice Research Newsletter (1983) Vol. 8, No. 6, pp. 16-17, reports pathogens and nematodes for the control of rice water weevil. Various strains of fungi (Beauveria bassiana and Metarrhizium anisopliae) and nematodes were shown to control rice water weevils. None of the B.t. biopreparations controlled rice water weevils when applied to 35 day old rice plants as a foliar spray at 0.6, 1.2 and 2.4 kg/ha.
It has been unexpectedly discovered that B.t. .delta.-endotoxins are effective in controlling rice water weevils contrary to published reports that B.t. biopreparations were ineffective in controlling this pest.